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Background Allele-specific DNA methylation (ASM) describes genomic loci that maintain CpG methylation at only one inherited allele rather than having coordinated methylation across both alleles. The most prominent of these regions are germline ASMs (gASMs) that control the expression of imprinted genes in a parent of origin-dependent manner and are associated with disease. However, our recent report reveals numerous ASMs at non-imprinted genes. These non-germline ASMs are dependent on DNA methyltransferase 1 (DNMT1) and strikingly show the feature of random, switchable monoallelic methylation patterns in the mouse genome. The significance of these ASMs to human health has not been explored. Due to their shared allelicity with gASMs, herein, we propose that non-traditional ASMs are sensitive to exposures in association with human disease. Results We first explore their conservancy in the human genome. Our data show that our putative non-germline ASMs were in conserved regions of the human genome and located adjacent to genes vital for neuronal development and maturation. We next tested the hypothesized vulnerability of these regions by exposing human embryonic kidney cell HEK293 with the neurotoxicant rotenone for 24 h. Indeed,14 genes adjacent to our identified regions were differentially expressed from RNA-sequencing. We analyzed the base-resolution methylation patterns of the predicted non-germline ASMs at two neurological genes, HCN2 and NEFM, with potential to increase the risk of neurodegeneration. Both regions were significantly hypomethylated in response to rotenone. Conclusions Our data indicate that non-germline ASMs seem conserved between mouse and human genomes, overlap important regulatory factor binding motifs, and regulate the expression of genes vital to neuronal function. These results support the notion that ASMs are sensitive to environmental factors such as rotenone and may alter the risk of neurological disease later in life by disrupting neuronal development.

Histone modification including H3 lysine 79 methylation (H3K79me) plays a key role during gene transcription and DNA damage repair. DOT1L, the sole methyltransferase for three states of H3K79me, is implicated in leukemia, co-lorectal cancer, and dilated cardiomyopathy. However, understanding of DOT1L and H3K79me in these pathways and disease pathogenesis has been limited due to the difficulty of working with DOT1L protein. For instance, locus-specific or genome-wide binding sites of DOT1L revealed by chromatin immunoprecipitation (ChIP)-based methods are necessary for inferring its functions, but high-quality ChIP-grade antibodies are currently not available. Herein we have developed a knock-in approach to tag endogenous DOT1L with 3 × Flag at its C-terminal domain to follow functional analyses. The knock-in was facilitated by using TALENs to induce a targeted double-strand break at the endogenous DOTIL to stimulate local homologous recombination at that site. The single cell colonies with successful knock-in were isolated and verified by different methods. We also demonstrated that tagged DOT1L maintains its normal function in terms of methylation and that the engineered cells would be very useful for further studies.

Nucleosome positioning and histone modification play a critical role in gene regulation, but their role during reprogramming has not been fully elucidated. Here, we determined the genome-wide nucleosome coverage and histone methylation occupancy in mouse embryonic fibroblasts (MEFs), induced pluripotent stem cells (iPSCs) and pre-iPSCs. We found that nucleosome occupancy increases in promoter regions and decreases in intergenic regions in pre-iPSCs, then recovers to an intermediate level in iPSCs. We also found that nucleosomes in pre-iPSCs are much more phased than those in MEFs and iPSCs. During reprogramming, nucleosome reorganization and histone methylation around transcription start sites (TSSs) are highly coordinated with distinctively transcriptional activities. Bivalent promoters gradually increase, while repressive promoters gradually decrease. High CpG (HCG) promoters of active genes are characterized by nucleosome depletion at TSSs, while low CpG (LCG) promoters exhibit the opposite characteristics. In addition, we show that vitamin C (VC) promotes reorganizations of canonical, H3K4me3- and H3K27me3-modified nucleosomes on specific genes during transition from pre-iPSCs to iPSCs. These data demonstrate that pre-iPSCs have a more open and phased chromatin architecture than that of MEFs and iPSCs. Finally, this study reveals the dynamics and critical roles of nucleosome positioning and chromatin organization in gene regulation during reprogramming.

Mutations in the PHF8 gene, which encodes the plant homeo domain (PHD) finger protein 8, are connected to X-linked mental retardation associated with cleft lip and cleft palate. Two groups now report that the PHF8 protein is a histone demethylase with activity against H4K20me1 (histone H4 lysine 20). Qi et al. report a role for PHF8 in regulating gene expression, as well as in neuronal cell survival and craniofacial development in zebrafish. The results suggest there may be a link between histone methylation dynamics and X-linked mental retardation. Liu et al. show that PHF8 is linked to two distinct events during cell-cycle progression. PHF8 is recruited to the promoters of G1/S-phase genes where it removes H4K20me1 and contributes to gene activation, whereas dissociation of PHF8 from chromatin in prophase allows H4K20me1 to accumulate during mitosis. PHF8 is a JmjC domain-containing protein, the gene for which has been linked to X-linked mental retardation (XLMR). These authors demonstrate PHF8 to be a histone demethylase with activity against H4K20me1. It has a role in regulating gene expression as well as in neuronal cell survival and craniofacial development in zebrafish. The results suggest there may be a link between histone methylation dynamics and XLMR. X-linked mental retardation (XLMR) is a complex human disease that causes intellectual disability1. Causal mutations have been found in approximately 90 X-linked genes2; however, molecular and biological functions of many of these genetically defined XLMR genes remain unknown. PHF8 (PHD (plant homeo domain) finger protein 8) is a JmjC domain-containing protein and its mutations have been found in patients with XLMR and craniofacial deformities. Here we provide multiple lines of evidence establishing PHF8 as the first mono-methyl histone H4 lysine 20 (H4K20me1) demethylase, with additional activities towards histone H3K9me1 and me2. PHF8 is located around the transcription start sites (TSS) of ∼7,000 RefSeq genes and in gene bodies and intergenic regions (non-TSS). PHF8 depletion resulted in upregulation of H4K20me1 and H3K9me1 at the TSS and H3K9me2 in the non-TSS sites, respectively, demonstrating differential substrate specificities at different target locations. PHF8 positively regulates gene expression, which is dependent on its H3K4me3-binding PHD and catalytic domains. Importantly, patient mutations significantly compromised PHF8 catalytic function. PHF8 regulates cell survival in the zebrafish brain and jaw development, thus providing a potentially relevant biological context for understanding the clinical symptoms associated with PHF8 patients. Lastly, genetic and molecular evidence supports a model whereby PHF8 regulates zebrafish neuronal cell survival and jaw development in part by directly regulating the expression of the homeodomain transcription factor MSX1/MSXB, which functions downstream of multiple signalling and developmental pathways3. Our findings indicate that an imbalance of histone methylation dynamics has a critical role in XLMR.

Imprinted genes are vulnerable to environmental influences during early embryonic development, thereby contributing to the onset of disease in adulthood. Monoallelic methylation at several germline imprints has been reported as DNMT1-dependent. However, which of these two epigenetic attributes, DNMT1-dependence or allelic methylation, renders imprinted genes susceptible to environmental stressors has not been determined. Herein, we developed a new approach, referred to as NORED, to identify 2468 DNMT1-dependent DNA methylation patterns in the mouse genome. We further developed an algorithm based on a genetic variation-independent approach (referred to as MethylMosaic) to detect 2487 regions with bimodal methylation patterns. Two approaches identified 207 regions, including known imprinted germline allele-specific methylation patterns (ASMs), that were both NORED and MethylMosaic regions. Examination of methylation in four independent mouse embryonic stem cell lines shows that two regions identified by both NORED and MethylMosaic (Hcn2 and Park7) did not display parent-of-origin-dependent allelic methylation. In these four F1 hybrid cell lines, genetic variation in Cast allele at Hcn2 locus introduces a transcription factor binding site for MTF-1 that may predispose Cast allelic hypomethylation in a reciprocal cross with either C57 or 129 strains. In contrast, each allele of Hcn2 ASM in J1 inbred cell line and Park7 ASM in four F1 hybrid cell lines seems to exhibit similar propensity to be either hypo- or hypermethylated, suggesting a ‘random, switchable’ ASM. Together with published results, our data on ASMs prompted us to propose a hypothesis of regional ‘autosomal chromosome inactivation (ACI)’ that may control a subset of autosomal genes. Therefore, our results open a new avenue to understand monoallelic methylation and provide a rich resource of candidate genes to examine in environmental and nutritional exposure models.

SummaryHistone modifications are implicated in influencing gene expression. We have generated high-resolution maps for the genome-wide distribution of 20 histone lysine and arginine methylations as well as histone variant H2A.Z, RNA polymerase II, and the insulator binding protein CTCF across the human genome using the Solexa 1G sequencing technology. Typical patterns of histone methylations exhibited at promoters, insulators, enhancers, and transcribed regions are identified. The monomethylations of H3K27, H3K9, H4K20, H3K79, and H2BK5 are all linked to gene activation, whereas trimethylations of H3K27, H3K9, and H3K79 are linked to repression. H2A.Z associates with functional regulatory elements, and CTCF marks boundaries of histone methylation domains. Chromosome banding patterns are correlated with unique patterns of histone modifications. Chromosome breakpoints detected in T cell cancers frequently reside in chromatin regions associated with H3K4 methylations. Our data provide new insights into the function of histone methylation and chromatin organization in genome function.

Nuclear factor erythroid-2 related factor 2 (Nrf2) is a vital transcription factor that regulates the anti-oxidative defense system. Previous reports suggested that the expression of the Nrf2 gene can be regulated by epigenetic modifications. The potential epigenetic effect of taxifolin (TAX), a potent cancer chemopreventive agent, in skin cancer chemoprotection is unknown. In this study, we investigated how Nrf2 is epigenetically regulated by TAX in JB6 P+ cells. TAX was found to inhibit the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced colony formation of JB6 P+ cells. TAX induced antioxidant response element (ARE)-luciferase activity in HepG2-C8 cells and up-regulated mRNA and protein levels of Nrf2 and its downstream genes heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase 1 (NQO1), in JB6 P+ cells. Furthermore, bisulfite genomic sequencing revealed that TAX treatment reduces the methylation level of the first 15 CpGs sites in the Nrf2 promoter. Western blotting showed that TAX inhibits the expression levels of DNA methyltransferase (DNMT) and histone deacetylase (HDAC) proteins. In summary, our results revealed that TAX can induce expression of Nrf2 and its downstream target genes in JB6 P+ cells by CpG demethylation. These finding suggest that TAX may exhibit a skin cancer preventive effect by activating Nrf2 via an epigenetic pathway.

PTIP in Immunoglobulin Switching One of the hallmarks of humoral immunity is the ability of immunoglobulins (Ig) to undergo class switch recombination (CSR). Through genetic recombining of the Ig heavy chain, Igs maintain their antigen specificity but gain the ability to interact with different cell surface receptors required for successful pathogen clearance. CSR requires transcription at the Ig heavy chain locus to initiate genetic rearrangement. Changes in chromatin accessibility are thought to promote CSR-associated transcription. Daniel et al. (p. 917 , published online 29 July; see the Perspective by Singh and Demarco ) now show that trimethylation of histone 3 at lysine 4 (H3K4me3) controls the accessibility of the Ig heavy chain locus to CSR and that PTIP (Pax interaction with transcription-activation domain protein-1), a component of the histone methylase complex, is required for this modification. Mouse PTIP-deficient B cells exhibited impaired CSR. PTIP was required both for the recruitment RNA Polymerase II and for subsequent chromatin remodeling, including histone acetylation, which occurs during CSR. Largely independent of its function in transcription initiation in CSR, PTIP also associated with double-stranded DNA breaks during CSR and promoted genome stability. These dual functions of PTIP may be important for the precise coordination of chromatin accessibility and recombination required during CSR.

SummaryMemory lymphocytes are characterized by their ability to exhibit a rapid response to the recall antigen, in which differential transcription is important, yet the underlying mechanism is not understood. We report here a genome-wide analysis of histone methylation on two histone H3 lysine residues (H3K4me3 and H3K27me3) and gene expression profiles in naive and memory CD8+ T cells. We found that specific correlation exists between gene expression and the amounts of H3K4me3 (positive correlation) and H3K27me3 (negative correlation) across the gene body. These correlations displayed four distinct modes (repressive, active, poised, and bivalent), reflecting different functions of these genes in CD8+ T cells. Furthermore, a permissive chromatin state of each gene was established by a combination of different histone modifications. Our findings reveal a complex regulation by histone methylation in differential gene expression and suggest that histone methylation may be responsible for memory CD8+ T cell function.

Histones are characterized by numerous posttranslational modifications that influence gene transcription1,2. However, because of the lack of global distribution data in higher eukaryotic systems3, the extent to which gene-specific combinatorial patterns of histone modifications exist remains to be determined. Here, we report the patterns derived from the analysis of 39 histone modifications in human CD4+ T cells. Our data indicate that a large number of patterns are associated with promoters and enhancers. In particular, we identify a common modification module consisting of 17 modifications detected at 3,286 promoters. These modifications tend to colocalize in the genome and correlate with each other at an individual nucleosome level. Genes associated with this module tend to have higher expression, and addition of more modifications to this module is associated with further increased expression. Our data suggest that these histone modifications may act cooperatively to prepare chromatin for transcriptional activation.

SummaryHistone acetyltransferases (HATs) and deacetylases (HDACs) function antagonistically to control histone acetylation. As acetylation is a histone mark for active transcription, HATs have been associated with active and HDACs with inactive genes. We describe here genome-wide mapping of HATs and HDACs binding on chromatin and find that both are found at active genes with acetylated histones. Our data provide evidence that HATs and HDACs are both targeted to transcribed regions of active genes by phosphorylated RNA Pol II. Furthermore, the majority of HDACs in the human genome function to reset chromatin by removing acetylation at active genes. Inactive genes that are primed by MLL-mediated histone H3K4 methylation are subject to a dynamic cycle of acetylation and deacetylation by transient HAT/HDAC binding, preventing Pol II from binding to these genes but poising them for future activation. Silent genes without any H3K4 methylation signal show no evidence of being bound by HDACs.

Background DNA methylation patterns are initiated by de novo DNA methyltransferases DNMT3a/3b adding methyl groups to CG dinucleotides in the hypomethylated genome of early embryos. These patterns are faithfully maintained by DNMT1 during DNA replication to ensure epigenetic inheritance across generations. However, this two-step model is based on limited data. Results We generated base-resolution DNA methylomes for a series of DNMT knockout embryonic stem cells, with deep coverage at highly repetitive elements. We show that DNMT1 and DNMT3a/3b activities work complementarily and simultaneously to establish symmetric CG methylation and CHH (H = A, T or C) methylation. DNMT3a/3b can add methyl groups to daughter strands after each cycle of DNA replication. We also observe an unexpected division of labor between DNMT1 and DNMT3a/3b in suppressing retrotransposon long terminal repeats and long interspersed elements, respectively. Our data suggest that mammalian cells use a specific CG density threshold to predetermine methylation levels in wild-type cells and the magnitude of methylation reduction in DNMT knockout cells. Only genes with low CG density can be induced or, surprisingly, suppressed in the hypomethylated genome. Lastly, we do not find any association between gene body methylation and transcriptional activity. Conclusions We show the concerted actions of DNMT enzymes in the establishment and maintenance of methylation patterns. The finding of distinct roles of DNMT1-dependent and -independent methylation patterns in genome stability and regulation of transcription provides new insights for understanding germ cell development, neuronal diversity, and transgenerational epigenetic inheritance and will help to develop next-generation DNMT inhibitors. Electronic supplementary material The online version of this article (doi:10.1186/s13059-015-0685-2) contains supplementary material, which is available to authorized users.

Background Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) has recently been used to identify the modification patterns for the methylation and acetylation of many different histone tails in genes and enhancers. Results We have extended the analysis of histone modifications to gene deserts, pericentromeres and subtelomeres. Using data from human CD4+ T cells, we have found that each of these non-genic regions has a particular profile of histone modifications that distinguish it from the other non-coding regions. Different methylation states of H4K20, H3K9 and H3K27 were found to be enriched in each region relative to the other regions. These findings indicate that non-genic regions of the genome are variable with respect to histone modification patterns, rather than being monolithic. We furthermore used consensus sequences for unassembled centromeres and telomeres to identify the significant histone modifications in these regions. Finally, we compared the modification patterns in non-genic regions to those at silent genes and genes with higher levels of expression. For all tested methylations with the exception of H3K27me3, the enrichment level of each modification state for silent genes is between that of non-genic regions and expressed genes. For H3K27me3, the highest levels are found in silent genes. Conclusion In addition to the histone modification pattern difference between euchromatin and heterochromatin regions, as is illustrated by the enrichment of H3K9me2/3 in non-genic regions while H3K9me1 is enriched at active genes; the chromatin modifications within non-genic (heterochromatin-like) regions (e.g. subtelomeres, pericentromeres and gene deserts) are also quite different.